灯盏细辛通过VDAC1/ROS/NLRP3通路减轻H_(2)O_(2)诱导的心肌细胞损伤  

Erigeron breviscapus attenuates H_(2)O_(2)-induced cardiomyocyte injury through VDAC1/ROS/NLRP3 pathway

作　　者：田源 肖雯 侯常苗 温慧莉 游三丽 袁李礼[1,2] TIAN Yuan;XIAO Wen;HOU Chang-miao;WEN Hui-li;YOU San-li;YUAN Li-li(Clinical Medicine School of Hunan University of Chinese Medicine,Changsha 410007,Hunan Province,China;Department of Cardiology,Brain Hospital of Hunan Province,Changsha 410007,Hunan Province,China;College of Life Science,Hunan Normal University,Changsha 410081,HunanPIrovince,China)

机构地区：[1]湖南中医药大学临床医学院,湖南长沙410007 [2]湖南省脑科医院心血管内科,湖南长沙410007 [3]湖南师范大学生命科学学院,湖南长沙410081

出　　处：《中国临床药理学杂志》2023年第20期2927-2931,共5页The Chinese Journal of Clinical Pharmacology

基　　金：湖南省中医药科研基金资助项目(D2022024);湖南省卫健委科研基金资助项目(202103010510);湖南省自然科学基金资助项目(2022JJ80062)。

摘　　要：目的 探讨灯盏细辛(EB)对过氧化氢(H_(2)O_(2))诱导的心肌细胞损伤的影响及其作用机制。方法 建立体外H_(2)O_(2)诱导的心肌细胞氧化应激损伤模型。将体外培养的HL-1小鼠心肌细胞分为空白组(常规培养)、对照组(4 mg·L^(-1) EB 1 h)、模型组(800μmol·L^(-1) H_(2)O_(2) 1 h)、实验组(4 mg·L^(-1) EB+800μmol·L^(-1) H_(2)O_(2) 1 h)。用细胞计数试剂盒-8(CCK-8)检测细胞活力;用乳酸脱氢酶(LDH)释放法检测细胞损伤情况;用流式细胞术检测活性氧(ROS)含量;用蛋白质印迹法检测核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、消皮素D(GSDMD)和电压依赖性阴离子通道1(VDAC1)蛋白表达水平;用酶联免疫吸附实验(ELISA)检测白细胞介素-1β(IL-1β)含量;用钙黄绿素乙酰甲酯(Calcein AM)检测线粒体通透性转换孔(mPTP)的开放程度。结果 空白组、对照组、模型组和实验组的细胞活力分别为(93.58±7.33)%、(95.18±4.24)%、(52.20±6.98)%和(74.80±5.33)%;LDH释放量分别为(44.18±6.36)、(46.16±5.01)、(689.60±124.00)和(365.60±87.79)U·L^(-1);ROS释放量分别为0.97±0.10、0.97±0.05、5.18±0.21和3.08±0.41;NLRP3蛋白表达水平分别为0.98±0.06、0.99±0.01、1.98±0.19和1.60±0.17;GSDMD蛋白表达水平分别为1.00±0.07、1.00±0.05、1.97±0.09和1.46±0.10;IL-1β释放量分别为(25.39±5.35)、(25.02±2.37)、(214.8±35.90)和(117.50±23.66)pg·mL^(-1);VDAC1蛋白表达水平分别为1.00±0.04、1.01±0.05、3.18±0.15和1.80±0.18;30 min的mPTP孔开放水平分别为1.22±0.02、1.22±0.01、1.14±0.02、1.17±0.01。模型组上述指标与空白组比较,差异均有统计学意义(P<0.05,P<0.01);实验组上述指标与模型组比较,差异均有统计学意义(P<0.05,P<0.01)。结论 灯盏细辛减轻H_(2)O_(2)诱导的HL-1心肌细胞损伤,其机制可能与通过VDAC1调节ROS/NLRP3通路有关。Objective To investigate the effect and mechanism o erigeron breviscapus (EB) on myocardial cell injury induced by hydrogen peroxide (H_(2)O_(2)).Methods The oxidative stress injury mode of cardiomyocytes induced by H_(2)O_(2)was established in vitro.The cultured mouse cardiomyocytes HL-1 were divided into 4 groups:blank group(normal culture),control group (4 mg·L^(-1)EB 1 h),model group(800μmol·L^(-1)H_(2)O_(2)1 h),experimental group (4 mg·L^(-1)EB+800μmol·L^(-1)H_(2)O_(2)1 h).Cell counting kit-8 (CCK-8) was used to detect the cell viability;the lactate dehydrogenase (LDH) release assay was used to detect cell damage;the flow cytometry was used to detect the content of reactive oxygen species (ROS);the Western Blot was used to detect the protein expression level of NOD-like receptor protein 3 (NLRP3),Gasdermin-D (GSDMD) and voltage-dependent anion channel 1 (VDAC1);the Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of interleukin-1β (IL-1β);the calcein methyl acetate (Calcein AM) was used to measure the openness of the mitochondrial permeability transition pore (m PTP).Results The cell viability of blank group,control group,model group and experimental group were (93.58±7.33)%,(95.18±4.24)%,(52.20±6.98)%and(74.80±5.33)%,respectively;the LDH release were (44.18±6.36),(46.16±5.01),(689.60±124.00) and(365.60±87.79) U·L^(-1),respectively;the ROS releases were 0.97±0.10,0.97±0.05,5.18±0.21 and3.08±0.41,respectively;the expression levels of NLRP3 protein were 0.98±0.06,0.99±0.01,1.98±0.19 and1.60±0.17,respectively;the expression levels of GSDMD protein were 1.00±0.07,1.00±0.05,1.97±0.09 and1.46±0.10,respectively;the contents of IL-1β were (25.39±5.35),(25.02±2.37),(214.8±35.90) and(117.50±23.66) pg·m L^(-1),respectively;the expression levels of VDAC1 protein were 1.00±0.04,1.01±0.05,3.18±0.15 and 1.80±0.18,respectively;the 30 min m PTP opening levels were 1.22±0.02,1.22±0.01,1.14±0.02 and 1.17±0.01,respectively.Compared with the blank group,the above

关 键 词：灯盏细辛 电压依赖性阴离子通道1 核苷酸结合寡聚化结构域样受体蛋白3 活性氧 焦亡 

分 类 号：R97[医药卫生—药品]